ULTRASTRUCTURAL IMMUNOCYTOCHEMISTRY WITH UNLABELED ANTIBODIES AND THE PEROXIDASE-ANTIPEROXIDASE COMPLEX A TECHNIQUE MORE SENSITIVE THAN RADIOIMMUNOASSAY

Abstract

Titration curves were developed with antisera to ^17-39ACTH (adrenocorticotropin) and ^1-39ACTH with the techniques of radioimmunoassay and electron microscopic immunocytochemistry. For the latter method, the unlabeled antibody peroxidase-antiperoxidase complex immunocytochemical technique was used to stain normal rat pituitary intermediate lobes. By radioimmunoassay standards, the ^17-39ACTH antiserum was of poor quality. Its titration curve exhibited a flat slope and it did not bind a significant amount of labeled antigen beyond a 1: 30 dilution. However, immunocytochemical staining was detected with this antiserum at dilutions as high as 1:1,500. The antiserum to ^1-39ACTH was of better quality by radioimmunoassay standards. It bound 45% of the labeled antigen at a dilution of 1:5,000. Immunocytochemical staining intensity was nearly maximal at a 1:5,000 dilution and decreased progressively to a limiting value at 1:16,000. However, when incubation times in the antisera were increased from 3 min to match those of the radioimmunoassay (48 hr) maximal staining was achieved at dilutions as great as 1:512,000 where only trace amounts of the labeled antigen were bound in the radioimmunoassay. It was concluded that the unlabeled antibody peroxidase-antiperoxidase complex immunocytochemical technique was sensitive enough to detect antibodies in sera of low titer and/or avidity which are not detected by a radioimmunoassay. The technique holds great promise for a sensitive assay system.